The formation and regulation of macromolecular complexes provides the backbone of aerobic energy production by the oxidative phosphorylation system within the mitochondrial membranes. Our parent grant is focused on cytochrome c oxidase (COX), the last mitochondrial respiratory chain enzyme, whose biogenesis involves a sophisticated network of dynamic protein-protein interactions that we are just beginning to disclose. COX is a heme a-copper-containing multimeric enzyme formed by polypeptides of nuclear and mitochondrial genetic origins. In the parent grant, we have revealed an assembly-feedback regulatory mechanism by which the synthesis of the key mtDNA-encoded and heme a-containing Cox1 subunit is regulated by the availability of its assembly partners in Saccharomyces cerevisiae. In this way, Cox1 synthesis and assembly are tightly coordinated. The central element of this regulatory system is the bi-functional COX1 mRNA translational activator and Cox1 chaperone Mss51 which is conserved from yeast to humans. During Cox1 synthesis, Mss51 interacts with the elongating polypeptide in translational complexes and in post-translational pre-assembly Cox1- containing high molecular weight complexes. These complexes are stabilized by two conserved small, single transmembrane domain proteins, Cox14 and the newly identified Cox25. At a point concurrent with Cox1 hemylation and/or the incorporation of subunit Cox5, Mss51 is released and is available for further rounds of translation. Cox14 and Cox25 instead, remain bound to Cox1 and other assembly chaperones to promote late stages of COX assembly. Recent observations suggest a similar mechanism exists in human cells. The parent grant explores the role of the human MSS51 homologue in COX assembly. Now, homologues of Cox14 and Cox25 have been also identified. The central hypothesis of this revision is the existence in human cells of an assembly-dependent regulation of Cox1 synthesis, in which MSS51, COX14 and COX25-containing multichaperone complexes act to coordinate Cox1 synthesis and multistep COX assembly. Two specific aims are proposed to characterize interacting partners of Cox14 and Cox25 and the conservation of these interactions from yeast to humans. Aim # 1 - Characterize MSS51, COX14 and COX25 knockout human cell lines created by a using a TALENs (transcription activator-like effector nucleases) approach. Aim # 2 - Characterize macromolecular multi-chaperone COX assembly complexes involving human MSS51, COX14 and COX25